7. PROJECT SUMMARY Millions of Americans suffer from sleep disturbances among which insomnia is the most common. Insomnia is often poorly treated with current pharmacotherapies and we propose to develop a new sleep-promoting therapeutic based on selective and direct activation of the G protein-gated inward rectifier K+ channels (GIRK). GIRK channels in the brain (predominantly GIRK1/2) play a key role in controlling neuronal excitability. Activation of the GIRK channels by neurotransmitters and neuropeptides, such as GABA and galanin, promote sleep. The link between GIRK function and animal NREM sleep was based on both preclinical and clinical evidence that shows GIRK activation by GABAB agonists, e.g., baclofen and ?-hydroxybutyric acid (GHB), preferentially promote non-rapid eye movement (NREM) sleep, leading to enhanced maintenance of sleep. In our collaborative project led by Dr. Weaver, we discovered the GIRK channel biologic probe ML297, which selectively activates recombinant human GIRK channels with a half-maximal effective concentration (EC50) of ~160 nM for neuronal GIRK1/2 with a ~4-fold selectivity over cardiac GIRK1/4 channels. Recently, we eliminated the concern of ML297 on cardiac safety using the cardiac ion channels panel consisting of hERG, hCav1.2 and hNav1.5 channels (IC50 =52, 22 and >30?M, respectively). ML297 had little effects on spontaneous action potential firing in hiPSC?derived cardiomyocytes upto 10M. Pharmacokinetic (PK) studies indicated that ML297 had acceptable brain penetration. Pharmacodynamic (PD) studies confirmed that ML297 can activate native neuronal GIRK channels in hypocretin/orexin neurons and hippocampal neurons. Furthermore ML297 (30?60 mg/kg, i.p.) inhibited spontaneous activity and prolonged inactive/sleep state in mice using our validated noninvasive wake/sleep monitoring system? SmartCageTM. A study employing electroencephalogram and electromyogram (EEG/EMG) recordings revealed that ML297 significantly enhanced NREM sleep in mice in a dose-dependent manner. We now propose to use ML297 as a starting point to develop a new therapeutic for insomnia because it is amenable to chemical modification to generate druggable analogs and it has no intellectual property roadblocks. We will achieve our goal through the accomplishment of two Specific Aims. Aim 1 Lead modification. Explore the structure-activity relationship (SAR) of ~200 new synthetic analogs derived from ML297 and identify the top 10 modified leads with increased potency and selectivity. Aim 2. Therapeutic lead discovery through in vitro and in vivo PK studies. We will determine the in vivo PK profiles of the top 3 new leads and test the best lead in a pilot sleep study in rats using EEG/EMG. Success in the Phase I project will demonstrate feasibility of discovering a druggable GIRK1/2 channel direct activator. In a Phase II project, we will focus on lead optimization to discover a safe therapeutic candidate with desirable PK/PD profile for the IND-enabling studies, and ultimately for the treatment of chronic insomnia.